The present invention relates to an improved rabies vaccine composition and a method for preparing same using viral-laden suckling mice or rat brain tissue.
Rabies vaccines are well known in the prior art and such vaccines are widely used in the treatment of both humans and animals. As is also well known, these vaccines, in order to be completely acceptable and fully effective, must produce or increase immunity to rabies with minimal side effects and the immunity imparted thereby must endure for a reasonably long period of time. Moreover, for these vaccines to be completely acceptable and fully effective, it is necessary that the same be a standardized preparation having a potency which remains relatively constant over reasonably long periods of time. Rabies virus can be grown in a number of tissues and tissue cultures.
In recent years, brains of suckling mice have been used as a suitable source for propagating rabies virus in the preparation of commercial animal vaccines, and the brains of suckling rats have been proposed (see, e.g., Lavender: Purified Rabies Vaccine (suckling rat brain origin) Appl. Microbiol. 19, (1970), pp. 923-927). Generally, in preparing rabies vaccines, a suspension of the viral-laden tissues in a buffered aqueous solution is prepared and then is inactivated. The pH-value of the buffered suspension and the inactivated vaccine composition is adjusted to a slightly basic value. A phosphate saline buffer solution is conventionally used for this purpose. However, several serious difficulties are encountered in conventional rabies vaccines containing a phosphate buffer:
(a) in order to maintain the desired slightly alkaline reaction and the buffering capacity of the phosphate buffer during the inactivation period, the pH-value of the suspension has to be repeatedly adjusted by adding potassium hydroxide solution; PA1 (b) the potency of the inactivated vaccine composition relative to the amount of viral-laden tissue is adversely affected by the presence of the phosphate buffer; and PA1 (c) the pH-value of the vaccine composition is stabilized only for a limited period of time after which the pH-value slowly decreases. This change towards an acid reaction causes serious problems in that acidity is detrimental to potency and is a false indication of bacterial contamination of the vaccine. Since contamination of a vaccine with bacteria usually leads to acidification of the vaccine composition, determining the reaction of a vaccine composition with the pH indicator agent phenol red is used as a simple method for detecting bacterial contamination of vaccine compositions. After a storage period of 9 to 14 months conventional phosphate buffer containing rabies vaccine compositions often react positive to the phenol red test, even though they are not contaminated at all. Due to this false indication of bacterial contamination large amounts of vaccines are unnecessarily discarded. PA1 (a) suspending a sufficient amount of viral laden suckling mice or rat brain tissue material in a sterilized aqueous buffer solution having a pH of between about 7.5 and about 8.4, and comprising an amount, dissolved therein, of between 0.03 and 0.08 moles per liter of buffer composition comprising a mixture of an organic base of the formula ##STR3## wherein R.sub.1 and R.sub.2 each are hydrogen or CH.sub.2 CH.sub.2 OH and an acid addition salt thereof with an acid the anion of which is compatible with virus replication, as to obtain a concentrated suspension comprising suspended therein an amount of at least about 20% by weight of proteineous viral laden suckling mice or rat brain tissue material; PA1 (b) comminuting the suspended viral laden suckling mice or rat brain material within the concentrated suspension into particles of injectable particle size; PA1 (c) diluting the concentrated suspension with a sufficient amount of said sterilized aqueous buffer solution to obtain a dilute suspension wherein the concentration of the proteineous viral laden brain particles is no greater than about 10% by weight; PA1 (d) inactivating the dilute suspension preferably by means of .beta.-propiolactone; and PA1 (e) adjusting the concentration of viral laden brain particles in the inactivated suspension to obtain the vaccine composition.